Complex host/symbiont integration of a multi-partner symbiotic system in the eusocial aphid Ceratovacuna japonica.

Some hemipteran insects rely on multiple endosymbionts for essential nutrients. However, the evolution of multi-partner symbiotic systems is not well-established. Here, we report a co-obligate symbiosis in the eusocial aphid, Ceratovacuna japonica. 16S rRNA amplicon sequencing unveiled co-infection with a novel Arsenophonus sp. symbiont and Buchnera aphidicola, a common obligate endosymbiont in aphids. Both symbionts were housed within distinct bacteriocytes and were maternally transmitted. The Buchnera and Arsenophonus symbionts had streamlined genomes of 432,286 bp and 853,149 bp, respectively, and exhibited metabolic complementarity in riboflavin and peptidoglycan synthesis pathways. These anatomical and genomic properties were similar to those of independently evolved multi-partner symbiotic systems, such as Buchnera-Serratia in Lachninae and Periphyllus aphids, representing remarkable parallelism. Furthermore, symbiont populations and bacteriome morphology differed between reproductive and soldier castes. Our study provides the first example of co-obligate symbiosis in Hormaphidinae and gives insight into the evolutionary genetics of this complex system.

Positive geographic correlation between soldiers' weapon size and defensive prowess in a eusocial aphid, Ceratovacuna japonica.

Some aphid species produce a soldier caste with enlarged forelegs and horns (weapons). It has been hypothesised that the evolution of morphological specialization by soldiers in social aphids is accelerated by high predation pressure, but this possibility has not been tested. Here, we investigated the relationship between local predator abundance and soldiers' weapon size and aggressiveness in a prey-predator system comprising a eusocial aphid, Ceratovacuna japonica, and its predators (larvae of the butterfly Taraka hamada and of the moth Atkinsonia ignipicta) in two populations with different predator abundances. We found that the soldiers in the predator-abundant population had larger weapons and were more aggressive than those in the population with lower predator abundance. Furthermore, the soldiers' defensive prowess (evaluated as the survival of aphids in the presence of predators) was greater in the predator-abundant population. These results provide the first evidence that a population of eusocial aphids experiencing high predation pressure has soldiers with pronounced defensive traits and defensive prowess.

Method for Measuring Bioactive Molecules in Blood by a Smartphone Using Bioluminescent Ratiometric Indicators.

Bioluminescent indicators facilitate determination of bioactive molecules in blood samples with high sensitivity. Using a bright luciferase, its bioluminescence (BL) can be easily detected by conventional light sensing devices. In this chapter, we describe a protocol to measure bioactive molecules in blood by taking the BL images with a smartphone camera. We exemplify the measurement of unconjugated bilirubin (UCBR) concentration in the blood of mice using a ratiometric bioluminescent UCBR indicator, BABI (bilirubin assessment with a bioluminescent indicator), and a smartphone camera. We show the UCBR concentration is easily determined through measuring the variance in the BL color with a smartphone camera. This method provides a practical method to lead to future point-of-care diagnosis with quick and simple procedures.

Application of Green-enhanced Nano-lantern as a bioluminescent ratiometric indicator for measurement of Arabidopsis thaliana root apoplastic fluid pH.

Plant root absorbs water and nutrients from the soil, and the root apoplastic fluid (AF) is an important intermediate between cells and the surrounding environment. The acid growth theory suggests that an acidic AF is needed for cell wall expansion during root growth. However, technical limitations have precluded the quantification of root apoplastic fluid pH (AF-pH). Here, we used Green-enhanced Nano-lantern (GeNL), a chimeric protein of the luciferase NanoLuc (Nluc) and the green fluorescent protein mNeonGreen (mNG), as a ratiometric pH indicator based on the pH dependency of bioluminescence resonance energy transfer efficiency from Nluc to mNG. Luminescence spectrum of GeNL changed reciprocally from pH 4.5 to 7.5, with a pKa of 5.5. By fusing GeNL to a novel signal peptide from Arabidopsis thaliana Cellulase 1, we localised GeNL in A. thaliana AF. We visualised AF dynamics at subcellular resolution over 30 min and determined flow velocity in the maturation zone to be 0.97± 0.06 µm/s. We confirmed that the developing root AF is acidic in the pH range of 5.1-5.7, suggesting that the AF-pH is tightly regulated during root elongation. These results support the acid growth theory and provide evidence for AF-pH maintenance despite changes in ambient pH.

Ratiometric Bioluminescent Indicator for a Simple and Rapid Measurement of Thrombin Activity Using a Smartphone.

Hemostasis is an essential function that repairs tissues and maintains the survival of living organisms. To prevent diseases caused by the abnormality of the blood coagulation mechanism, it is important to carry out a blood test periodically by a method that is convenient and less burdensome for examiners. Thrombin is a protease that catalyzes the conversion of fibrinogen, and its cleavage activity can be an index of coagulation activity. Here, we developed a ratiometric bioluminescent indicator, Thrombastor (thrombin activity sensing indicator), which reflects the thrombin cleavage activity in blood by changing the emission color from green to blue. Compared to the current thrombin activity indicator, the rapid color change of the emission indicated a 2.5-fold decrease in the Km for thrombin, and the cleavage rate was more than two times faster. By improving the absolute bioluminescence intensity, detection from a small amount of plasma could be achieved with a smartphone camera. Using Thrombastor and a versatile device, an effective diagnosis for preventing coagulation disorders can be provided.

Method for Detecting Emission Spectral Change of Bioluminescent Ratiometric Indicators by a Smartphone.

The application of smartphones as detectors is essential to achieve ubiquitous measurement targeting biomolecules. Because bioluminescence (BL), as a tag for a target sample, does not require an excitation light source, it can be combined with a smartphone to constitute a compact and mobile measurement system. A method was recently established to detect the spectral change of ratiometric indicators based on bioluminescence resonance energy transfer with a smartphone camera. For example, it was possible to detect changes in the BL color of the Ca2+ indicator quantitatively and easily calculate the concentration of free Ca2+ by setting appropriate image acquisition conditions in a smartphone application. In this paper, we describe techniques to obtain scientifically relevant and reliable BL data with such a convenient instrument. This protocol expands the potential of the smartphone as a personal imaging device with high mobility that can be used anywhere.

Intraspecific convergence of floral size correlates with pollinator size on different mountains: a case study of a bumblebee-pollinated Lamium (Lamiaceae) flowers in Japan.

BACKGROUND: Geographic differences in floral size sometimes reflect geographic differences in pollinator size. However, we know little about whether this floral size specialization to the regional pollinator size occurred independently at many places or occurred once and then spread across the distribution range of the plant species. RESULTS: We investigated the relationship between the local floral size of flowers and local pollinator size in 12 populations of Lamium album var. barbatum on two different mountains in the Japan Alps. Then, using 10 microsatellite markers, we analyzed genetic differentiation among the 12 populations. The results showed that local floral size was correlated with the average size of relevant morphological traits of the local pollinators: floral size was greater in populations visited frequently by the largest flower visitors, Bombus consobrinus queens, than it was in other populations. We also found that the degree of genetic similarity between populations more closely reflected interpopulation geographic proximity than interpopulation similarity in floral size. CONCLUSIONS: Although genetic similarity of populations was highly associated with geographic proximity, floral size varied independently of geographic proximity and was associated with local pollinator size. These results suggest that in L. album var. barbatum, large floral size evolved independently in populations on different mountains as a convergent adaptation to locally abundant large bumblebee species.

Ratiometric Bioluminescent Indicator for Simple and Rapid Diagnosis of Bilirubin.

Bilirubin in human blood is highly important as a general index of one's physical condition because its concentration changes under the influence of several diseases. In particular, in newborns, jaundice is one of the most common diseases involving unconjugated bilirubin (UCBR), causing serious symptoms such as nuclear jaundice and deafness. Therefore, a frequent measurement of the UCBR levels in the blood is important. Here, we report a ratiometric bioluminescent indicator, BABI (bilirubin assessment with a bioluminescent indicator), that changes the emission color from blue to green depending on the UCBR concentration in a sample. Owing to the use of a bioluminescence signal that has a higher signal-to-noise ratio than the absorption and fluorescence signal, BABI enables highly sensitive and quantitative detection of UCBR for small blood samples using a smartphone camera. The establishment of a UCBR measurement assay using BABI provides the possibility of a simple and rapid method for blood-based diagnosis using bioluminescent indicators and a versatile mobile device.

Smartphone-Based Portable Bioluminescence Imaging System Enabling Observation at Various Scales from Whole Mouse Body to Organelle.

Current smartphones equipped with high-sensitivity and high-resolution sensors in the camera can respond to the needs of low-light imaging, streaming acquisition, targets of various scales, etc. Therefore, a smartphone has great potential as an imaging device even in the scientific field and has already been introduced into biomolecular imaging using fluorescence tags. However, owing to the necessity of an excitation light source, fluorescence methods impair its mobility. Bioluminescence does not require illumination; therefore, imaging with a smartphone camera is compact and requires minimal devices, thus making it suitable for personal and portable imaging devices. Here, we report smartphone-based methods to observe biological targets in various scales using bioluminescence. In particular, we demonstrate, for the first time, that bioluminescence can be observed in an organelle in a single living cell using a smartphone camera by attaching a detachable objective lens. Through capturing color changes with the camera, changes in the amount of target molecules was detected using bioluminescent indicators. The combination of bioluminescence and a mobile phone makes possible a compact imaging system without an external light source and expands the potential of portable devices.

Seasonal Migration in the Aphid Genus Stomaphis (Hemiptera: Aphididae): Discovery of Host Alternation Between Woody Plants in Subfamily Lachninae.

About 10% of aphid species show host alternation. These aphids migrate between primary and secondary host plant species in spring and autumn. Host alternation has not been observed in subfamily Lachninae, although it has been suggested on the basis of circumstantial evidence that Stomaphis japonica (Takahashi) may alternate its host between Quercus serrata (Murray) and Quercus acutissima (Carruth). However, a molecular phylogenetic study has indicated that the Stomaphis individuals feeding on these two plant species belong to two different lineages and aphids feeding on Q. acutissima and Pinus densiflora (Sieb. & Zucc.) belong to the same lineage. Here, we examined host alternation in Stomaphis species by comparing molecular phylogenetic identities, morphological features, and life cycles. The molecular analysis and morphological examination showed that aphids feeding on Q. acutissima were the same as those feeding on P. densiflora, whereas aphids feeding on Q. serrata were different from those feeding on Q. acutissima or on P. densiflora. Furthermore, winged aphids were observed on both Q. acutissima and P. densiflora in autumn, but we did not observe winged aphids on Q. serrata. These results indicate that Stomaphis (Walker) individuals feeding on Q. serrata and Q. acutissima belong to two species, one that feeds year-round on Q. serrata, and another, heteroecious species that feeds on P. densiflora as a primary host and on Q. acutissima as a secondary host. This study documents host alternation in subfamily Lachninae for the first time and discusses the acquisition of host alternation by Stomaphis from evolutionary and ecological perspectives.

Bioluminescent Ratiometric Indicator for Analysis of Water Hardness in Household Water.

Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca2+/Mg2+ detection domain of human centrin 3. The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.

Evolutionary diversification of Japanese Stomaphis aphids (Aphididae, Lachninae) in relation to their host plant use and ant association.

Phytophagous insects are among the most diverse of the earth's organisms, and their diversification patterns and the driving forces behind these have attracted considerable research interest. Host shifting to closely related plant species is thought to play an important role in phytophagous insect diversification, but the extent to which other interactions such as mutualistic associations affect diversification is not yet known. In this study, we reconstructed the molecular phylogeny of Japanese Stomaphis aphids and determined whether host shifting or mutualistic association with different ant species could explain diversification in this aphid genus. We analyzed 12 species of Stomaphis and grouped them into ten well-supported DNA lineages. Species in each lineage used a single or a few host plant species, but were mutualistically associated with many ant species of the genus Lasius. This result suggests that Stomaphis evolutionarily diversified primarily through host plant shifts. Interestingly, the reconstructed phylogeny suggests that Stomaphis host shifts occasionally occurred between very distantly related host plant taxa (spanning up to five plant orders). The dependence of Stomaphis on long-lasting Lasius ant colonies situated in temperate deciduous forests where Lasius is the dominant ant genus may have led the aphids to shift to distantly related but spatially adjacent host tree species.

A simple microfluidic device for live-imaging of the vertical section of epithelial cells.

We investigated the capability of simple microfluidic devices with trenches having vertical sidewalls for live-cell fluorescence imaging of adherent cells. An epithelial cell line that forms a two-dimensional (2D) sheet was cultured to adhere to the vertical sidewall so that its vertical section can be imaged directly using ordinal inverted-type laser-scanning microscopy. The material and the structure of the device were characterized. We show that the detailed distribution of intracellular organelles, such as microtubules and mitochondria, and of intercellular apparatus, such as claudin and zonula occludens, can be imaged with high spatio-temporal resolution with a single scan.

Cooperative interaction among BMAL1, HSF1, and p53 protects mammalian cells from UV stress.

The circadian clock allows physiological systems to adapt to their changing environment by synchronizing their timings in response to external stimuli. Previously, we reported clock-controlled adaptive responses to heat-shock and oxidative stress and showed how the circadian clock interacts with BMAL1 and HSF1. Here, we present a similar clock-controlled adaptation to UV damage. In response to UV irradiation, HSF1 and tumor suppressor p53 regulate the expression of the clock gene Per2 in a time-dependent manner. UV irradiation first activates the HSF1 pathway, which subsequently activates the p53 pathway. Importantly, BMAL1 regulates both HSF1 and p53 through the BMAL1-HSF1 interaction to synchronize the cellular clock. Based on these findings and transcriptome analysis, we propose that the circadian clock protects cells against the UV stress through sequential and hierarchical interactions between the circadian clock, the heat shock response, and a tumor suppressive mechanism.

A genetic screen to discover SUMOylated proteins in living mammalian cells.

Post-translational modification by the Small Ubiquitin-related Modifier (SUMO) is indispensable for diverse biological mechanisms. Although various attempts have been made to discover novel SUMO substrate proteins to unveil the roles of SUMOylation, the reversibility of SUMOylation, and the differences in the SUMOylation level still makes it difficult to explore infrequently-SUMOylated proteins in mammalian cells. Here, we developed a method to screen for mammalian SUMOylated proteins using the reconstitution of split fluorescent protein fragments in living mammalian cells. Briefly, the cells harboring cDNAs of SUMOylated proteins were identified by the reconstituted fluorescence emission and separated by cell sorting. The method successfully identified 36 unreported SUMO2-substrate candidates with distinct intracellular localizations and functions. Of the candidates, we found Atac2, a histone acetyltransferase, was SUMOylated at a lysine 408, and further modified by multiple SUMOs without isoform specificity. Because the present method is applicable to other SUMO isoforms and mammalian cell-types, it could contribute to a deeper understanding of the role of SUMOylation in various biological contexts.

Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres.

Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function.

Bioluminescent Indicator for Highly Sensitive Analysis of Estrogenic Activity in a Cell-Based Format.

Estrogens regulate different physiological systems with wide ranges of concentrations. The rapid analysis of estrogens is crucially important for drug discovery and medical diagnosis, but quantitation of nanomolar estrogens in live cells persists as an important challenge. We herein describe a bioluminescent indicator used to detect low concentrations of estrogens quantitatively with a high signal-to-background ratio. The indicator comprises a ligand-binding domain of an estrogen receptor connected with its binding peptide, which is sandwiched between split fragments of a luciferase mutant. Results show that the indicator recovered its bioluminescence upon binding to 17ß-estradiol at concentrations higher than 1.0 × 10-10 M. The indicator was reactive to agonists but did not respond to antagonists. The indicator is expected to be applicable for rapid screening estrogenic compounds and inhibitors, facilitating the discovery of drug candidates in a high-throughput manner.

Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis.

G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and ß-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

Confocal Bioluminescence Imaging for Living Tissues with a Caged Substrate of Luciferin.

Fluorescence imaging can elucidate morphological organization and coordinal networks, but its background luminescence degrades the image contrast. Our confocal bioluminescence imaging system uses a luciferase caged substrate, with light passing through multipinhole arrays, causing bioluminescence at a focal plane. After a charge-coupled device camera captures luminescence, the imaging system acquires confocal images of multilayered cells with depth information, supporting quantitative analysis of spatial cellular localization in living tissues.